Abstract
The process of decellularization is crucial for producing a substitute for the absent tracheal segment, and the choice of agents and methods significantly influences the outcomes. This paper aims to systematically review the efficacy of diverse tracheal decellularization agents and methods using the PRISMA flowchart. Inclusion criteria encompassed experimental studies published between 2018 and 2023, written in English, and detailing outcomes related to histopathological anatomy, DNA quantification, ECM evaluation, and biomechanical characteristics. Exclusion criteria involved studies related to 3D printing, biomaterials, and partial decellularization. A comprehensive search on PubMed, NCBI, and ScienceDirect yielded 17 relevant literatures. The integration of various agents and methods has proven effective in the process of tracheal decellularization, highlighting the distinct advantages and drawbacks associated with each agent and method.
1. Introduction
Trachea’s abnormality has been a rising problem in recent years, both congenital abnormalities and acquired abnormalities. Congenital defects may vary in degree and forms, including tracheal agenesis, massive tracheo-oesophageal fistula, and/or tracheomalacia. Acquired defects are commonly caused by trauma, be it blunt or sharp. According to literature, the worst and the most common case of traumatic tracheal abnormalities is post intubation tracheal stenosis (PITS), with the prevalence of 6%–21% of intubated patients. Around 10% of mild stenosis patients may remain undetected for more than 10 years. The newest literature shows that the prevalence of PITS in London is 926 new cases per year [1].
There are 2 types of tracheal repair based on the affected segments. In short-segment defect, which is defined as defects in less than half of the total tracheal length in adults and/or less than a third of the total tracheal length in children, the go-to procedure would be either an end-to-end tracheoplasty or a slide tracheoplasty [2]. On the other hand, long-segment defect is defined as defects in half or more of the total tracheal length in adults and/or a third or more of the total tracheal length in children. There are still no definitive treatments for long segment defects. The patient would usually receive temporary palliative care, such as T-tubes or stents, which have a high rehospitalization and infection prevalence [2].
In recent years, the field of regeneration medicine has been trying to develop a definitive treatment for long-segment tracheal defects and the most promising method right now would be replacing the affected part. Tracheal grafts can be divided into groups such as autologous, homograft, prosthetic, and combined graft. Each of the groups has their own strengths and weaknesses. In autologous and homografts, the problem lies in finding a donor, vascularization failure, failure in thriving, and the need for prolonged immunosuppressants. Prosthetic grafts on the other hand may be a solution to some of those problems. However, prosthetic grafts are too inflexible and proinflammatory, making them the second-best option for a tracheal graft [3].
In order to find a suitable tracheal replacement, graft materials development has been increasing these past few years. Tissue engineering has been used in various ways, such as blood vessels and heart valves. Tissue engineering needs 3 major components, which are scaffolds, healthy cells, and a bioreactor.
Scaffolds are the base of the new cells. Scaffold should have the same mechanical properties of the original organs and should be able to support the cells’ adhesion, migration, proliferation, and differentiation. According to these standards, the best tracheal scaffold right now would be a decellularized trachea as it has the same biomechanics, flexibility, and proangiogenic. The purpose of decellularization is to remove the immunogenic components of the hosts without harming the extracellular matrices (ECMs) since ECMs’ main function is to grow, maintain, and regenerate the cells. ECM’s main components are collagen, proteoglycan, glycoprotein, and glycosaminoglycan. There are 3 methods of decellularization, which are enzymatic, chemical, and physical. The most popular one is the chemical decellularization method where detergent is used to decellularize the scaffold. In tracheal decellularization, the tricky part is the cartilage as it is really thick and will take time for the detergent to penetrate; meanwhile, the ECMs cannot take that much detergent exposure. Thus, this paper is written to evaluate the various decellularization methods and to find one that removes the most immunogenic components yet preserve the most ECMs [4, 5].
The objective of this research is to assess the effectiveness of various tracheal decellularization methods in experimental animals’ trachea by the deoxyribonucleic acid (DNA) count and histopathological analysis (HPA) examinations.
2. Materials and Methods
The inclusion criteria for this research were animal experimental studies published between the years 2018 and 2023, written in English, primarily studying the process of tracheal decellularization, and describing the outcomes of histopathological analysis, DNA, ECM, and biomechanical characteristics. Excluded studies were studies aiming for partial decellularization, having end products of nontracheal grafts, and/or involving 3D printing and biomaterials. The search was done through PubMed, NCBI, and ScienceDirect using the terms trachea or tracheal and decellularization or decellularization. The last search was done in September 2023. The population, intervention, comparison, and outcome (PICO) framework was used as described in Table 1. The detail of the literature identification process was explained using the PRISMA flowchart in Figure 1.
3. Results and Discussion
A comprehensive search of PubMed, NCBI, and ScienceDirect yielded 1044 potentially eligible studies. Of these, 200 duplicates were removed. Upon title and abstract screening, 808 studies were excluded due to being reviews, case reports, editorial comments, not in English, or unrelated to the topic. Two studies were disqualified due to unavailability of full texts. Subsequently, a meticulous full-text review led to the exclusion of 16 more studies, including those with partial decellularization, improper outcomes, incorrect interventions, and nonexperimental designs. Consequently, a total of 17 studies were selected for inclusion in this review. The characteristics of the methods, agents used, and outcomes of each included studies are presented inTable 2.
3.1. Tracheal Anatomical Properties
Structurally, the tracheobronchial system can be classified into the conductive part (cartilage) and the airway part (noncartilage). It is located in the medial side of the body where it extends from the neck and to the thorax; topographically, it starts from vertebrae C5-6 and extends down until T5 where it will branch into 2 bronchi. It connects the larynx and the bronchus, and functionally, it is semiflexible, 1.5–2 cm wide, and 10−13 cm long [3, 22].
Tracheal structure includes mucosa, submucosa, hyaline cartilage, and adventitial layer. The mucosal layer includes pseudostratified columnar epithelium and goblet cells; goblet cells will secrete mucous to trap the debris and dirt for the cilia will sweep them away. Submucosa is the deepest part of the tracheal lumen with the most blood vessels and nerve, and its function is to maintain tracheal structural integrity [3, 22].
3.2. Tracheal ECM Roles
Tracheal biomechanical properties come from the ECMs which consist of glycosaminoglycan (GAG), collagen, proteoglycan, and other glycoproteins. Collagen is the main component that gives the trachea its biomechanical properties. The collagen fibers make the trachea characteristically laterally rigid and longitudinally flexible. In addition, the ECMs have three main functions, which are intercellular signalling via paracrine signalling, intracellular signalling via autocrine signalling, and cellular formation via mechanical pressure [3, 22, 23].
3.3. Tracheal Tissue Engineering
Tracheal tissue engineering has been on the rise due to the complications of autografts and allografts. Tracheal tissue engineering includes resecting the affected organ and changing it with a scaffold that has been seeded with stem cells. The main components of tracheal tissue engineering are the scaffold, cell source, agent, and method [3, 22, 23].
3.4. Tracheal Scaffold
There are two main types of tracheal scaffolds with their own strength and weaknesses. The first is the synthetic tracheal scaffold. It is more versatile when it comes to shape and size, but the macro- and microanatomy of the scaffold is lacking compared to the biological scaffold. One of the examples of the synthetic tracheal scaffold includes biodegraded molecules from polyglycolic acid and nanocomposite polymer (POSS) covalently bonded to polyurethane (PCU) [24].
The second type is the biological decellularized scaffold. This type is more popular and favourable since it supports the cellular adhesion, proliferation, and differentiation process [25]. The decellularization process is needed in order to lose all the immune-inducing systems within the trachea that can be activated with major histocompatibility complex I and II (MHC-I and MHC-II) [23]. The components of the natural decellularized scaffolds are exactly like the original. The only downside is that during the decellularization process, there might be some cellular and structural changes due to ECM destruction. Therefore, some researchers are looking for a way to minimize the ECM destruction while optimizing the decellularization process. One of the advantages of using bioscaffold is that the patients have no need of taking immunosuppressants since the tracheas are decellularized and seeded with the patient’s stem cells [22].
3.5. Decellularization Process
Decellularization is the act of eliminating immunogenic cells without damaging the ECMs; ECMs here refer to structural protein (collagen and elastin), special protein (fibrillin, fibronectin, and laminin), proteoglycan (heparin sulfate, chondroitin sulfate, keratin sulfate, and GAG), and growth factors. The advantages of decellularized tracheas are less antigenicity, inflammation, and graft rejection [26].
Every decellularization process needs a decellularization agent and every decellularization agent has its own pros and cons. Some of the popular decellularization agents used are mentioned in the following.
3.5.1. Chemical Agent
The types of chemical agents commonly used include acids, bases, detergents, hypotonic-hypertonic solutions, and solvents to lyse and kill cells [26, 27]. Some of the chemicals used in the decellularization process are as follows.
(1) Acid and Bases. Decellularization methods involving acids and bases catalyse the hydrolytic degradation of biomolecules, cytoplasmic components, and nucleic acids [28]. Like detergents, they have the capacity to disrupt the extracellular matrix (ECM) constituents and structures. Acidic compounds either donate hydrogen ions (H+) or form covalent bonds with electron pairs to facilitate hydrolytic degradation. Peracetic acid (PAA), hydrochloric acid, and acetic acid are commonly employed for the decellularization process [29, 30].
Peracetic acid and hydrochloric acid are among the acid agents employed in the decellularization process. Peracetic acid functions by disrupting cell membranes and solubilizing cytoplasmic organelles. However, it comes with the drawback of damaging the extracellular matrix (ECM) architecture. On the other hand, hydrochloric acid induces cell lysis, denatures proteins, and catalyses the hydrolytic degradation of biomolecules. Yet, its disadvantage lies in its impact on intracellular molecules, particularly glycosaminoglycans (GAG) [31–33]. Hence, it is essential to choose suitable acids and concentrations. Peracetic acid (PAA) at 0.1% concentration is considered an optimal treatment for thin tissues, as it minimally affects extracellular matrix (ECM) structures and components [34].
In contrast to acidic compounds, alkaline substances can release hydroxide ions (OH−) and interact with acids to produce salts. Ammonium hydroxide, sodium hydroxide, and sodium sulphide are commonly used bases in decellularization [35]. Bases achieve tissue decellularization by denaturing chromosomal DNA and inducing cellular lysis. Particularly, alkaline solutions with a pH exceeding 11 prove effective in eliminating cellular remnants, given the susceptibility of DNA to denaturation [34].
Ammonium hydroxide functions by solubilizing cytoplasmic components, disrupting nucleic acids, and catalysing the hydrolytic degradation of biomolecules. However, drawbacks include its impact on the GAG content, collagen, and growth factors, as well as a weakening of the mechanical properties of the scaffold. Alkaline solutions with a pH range of 10–12 can cause significant harm to collagen fibers, fibronectin, and GAGs. In addition, they may trigger intense host responses and lead to the formation of fibrotic tissues [32, 35, 36].
(2) Organic Diluent. The mechanism of action of these agents involves cell membrane lysis. Commonly utilized types include alcohol, acetone, and 1% tributyl phosphate (TBP) for solid tissue decellularization. In the case of acetone and alcohol, they have the capacity to precipitate ECM proteins and influence ECM ultrastructure. In the decellularization of solid tissues such as tendons, tributyl phosphate is more effective at preserving ECM structure and composition. In addition, TBP demonstrates virucidal effects (inactivating viruses) without affecting coagulation factors [26, 27].
(3) Hyper/Hypotonic Fluid. Hypotonic solutions lyse cell membranes by increasing cell volume beyond their limits. These agents do not significantly impact changes in ECM components. Hypertonic solutions cause cells to lose volume and eventually die. The drawback of this type is its incapacity to effectively remove residual DNA from cell death. In the process, scaffold materials are immersed in hypotonic and/or hypertonic solutions over several cycles to achieve optimal results [26, 27].
(4) Ionic Detergent. Ionic detergent includes sodium dodecyl sulfate (SDS), sodium dodecyl cholate (SDC), Triton X-200, and sodium hypochlorite. The most popular one right now is the SDS 0.1%. Sodium dodecyl sulfate (SDS), also known as sodium lauryl sulfate (SLS), is a widely recognized anionic surfactant with amphiphilic properties, combining hydrophilic and hydrophobic characteristics. It dissolves lipids but can cause skin and eye irritation. SDS is produced by reacting dodecanol with sulfuric acid and sodium hydroxide, and the chemical reaction produced is as follows (Figure 2) [37, 38].
SDS 0.1% is a commonly used agent, but various concentrations of SDS (0.01, 0.025, 0.05, 0.075, and 0.1%) have started to be studied in the context of the decellularization process. Like nonionic detergents, this agent can also influence the ECM structure. Furthermore, this agent can remove growth factors in the ECM [26, 27].
(5) Nonionic Detergent. Nonionic detergent will disrupt the cellular structure by destroying the lipid-lipid and lipid-protein bind without destroying the protein-protein compound. Triton X-100 0.5% is the most popular one due to its ability to maintain protein structures and GAG sulfate. The advantage of these agents is that they do not disrupt the bonds between proteins or sulfated glycosaminoglycans. Their drawback, however, is their potential to reduce the concentration of laminin/fibronectin in the ECM structure [26, 27].
(6) Zwitterionic Detergent. This agent has the combined properties of ionic and nonionic detergent. This agent is usually used for mild-moderate decellularization. Some of the examples include sulfobetaine-10 (SB10), sulfobetaine-16 (SB16), and 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) [26, 27].
3.5.2. Physical Agent
Physical agents employ various physical conditions, including temperature, mechanical force, pressure, and electrical currents, to disrupt cell membranes and induce lysis, ultimately resulting in the removal of cells from the scaffold matrix. These physical methods represent an alternative approach to decellularization and have been explored for their potential benefits in tissue engineering [26, 27].
(1) Freezing-Thawing. Decellularization is done by purposefully freezing the intracellular fluid and destroying the cells; the downsides of this method are that it cannot get rid of the genetic materials and ruins the ECMs. This agent is usually done with the help of some detergents and/or nuclease to optimize the results [26, 27].
(2) Mechanical Pressure. These agents are typically employed in organs or tissues with less dense ECM (e.g., the lungs and the liver). Their mechanism of action involves releasing cells from the tissue or organ through applied pressure. However, one drawback of these agents is their potential to cause structural damage to the ECM. Precisely controlling the applied force is a crucial aspect of using these agents effectively [26, 27].
(3) Electroporation. Another term for this agent is nonthermal irreversible electroporation. Its mechanism involves disrupting the potential difference across the cell membrane, thereby interfering with cell permeability and ultimately causing cell death using an electric current. An advantage of this agent is its ability to preserve the biomechanical structure of the ECM. However, a limitation is its suitability for use in small, thin tissues or smaller organs [26, 27].
(4) Immersion and Agitation. Immersion and agitation represent a more suitable approach for small, delicate, and thin organ sections, as well as tissues lacking intrinsic vascular structures [39–41]. Immersion and agitation involve submerging tissues in decellularization solutions with continuous mechanical agitation, and its effectiveness relies on various parameters such as agitation intensity, decellularization agent, and tissue dimensions [39, 42]. The process, following tissue immersion in the agents, facilitates cell rupture, cell detachment from basement membranes, and the elimination of cellular components. Employing immersion and agitation as an optimal physical decellularization method offers numerous advantages. First, dynamic immersion and agitation achieve a more uniform detergent exposure compared to static decellularization, resulting in better decellularization outcomes with reduced exposure time to aggressive agents [43, 44]. Second, this method minimally impacts ECM surface structure, collagen integrity, mechanical strength, and GAG content [44–48]. Third, it is more accessible and easily executed than whole organ perfusion. However, it may inflict more tissue damage compared to perfusion due to the limited chemical diffusion caused by agitation [34].
(5) Sonication. Sonication operates by generating acoustic cavitation bubbles, inducing shear stress effects, and consequently rupturing the cell membrane. It facilitates agent penetration by emitting vibrations, aiding in the removal of cellular debris. However, the drawback lies in the potential disruption of main structural fibers and adverse effects on vascular tissues with high power or prolonged duration of sonication [49–51].
3.5.3. Enzymatic Agent
(1) Trypsin. The working mechanism of this agent involves breaking the peptide bonds between carboxyl, arginine, and lysine. Several studies using 0.5% and 1% trypsin with exposure times of 48 and 24 hours have been shown to cause ECM damage. Further research on 0.02% trypsin for 1 hour has a less significant impact on the ECM structure after decellularization [26, 27].
(2) Exo/Endonuclease. The working mechanism of this agent involves breaking the bonds of RNA and DNA components. Commonly used types of this agent include DNase (0.2–0.5 mg/mL) and RNase (0.2–50 μg/mL). The application of this agent is often combined with others to remove any remaining DNA/RNA from the decellularized scaffold [26, 27].
(3) Dispase. The mechanism of action of this agent involves catalysing primarily collagen IV and fibronectin in the basement membrane, separating it from the epithelial layer. Commonly used types of this agent include 4 mg/mL Dispase II for 45 minutes to remove it. For the removal of hair, fat, and epidermis, Dispase II at 0.24 mg/mL for 3 hours is used [26, 27].
(4) Phospholipase A2. The mechanism of action of this agent involves damaging phospholipid components. This agent is typically used in combination with other decellularization agents. Its advantage is in preserving collagen and proteoglycans in the ECM structure although it has a minor impact on GAG composition [26, 27].
3.5.4. Compound Agent
Physical, chemical, and enzymatic agents each have their own advantages and disadvantages and work through distinct mechanisms. To make the decellularization process more effective and efficient, the use of combinations of agents has been explored. For example, a combination of physical and chemical methods has led to the development of cryochemical agents for liver decellularization. Another example involves the combination of agitation, alkali, detergent, enzymes, and light-emitting diodes in the decellularization process of tracheal organs [26, 27].
3.6. Decellularization Methods
After determining the decellularization agent, the next step would be choosing the method. The organ’s or tissue’s characteristics need to be considered in choosing the decellularization method. Some of the examples include the following.
3.6.1. Whole Organ Perfusion
It is used on large and dense organs or tissues with internal vascularization and usually uses ante- or retrograde perfusion. This method uses the organ’s own vascular system to distribute the decellularization agent; after distributing the decellularization agent, the dead cells and the remaining decellularization agent will be drained through the veins. Some of the organs that can be decellularized through this method are the muscle, lungs, liver, kidneys, and heart [26, 27].
3.6.2. Immersion and Agitation
This method is used for organs without any decent internal vascularization; the tissue would be immersed and agitated in the decellularization agent where the agent will diffuse into the cells. The factors that affect the outcome include the agitation intensity, decellularization agent, and tissue density and size. It usually takes 1-2 hours for thin preparations and 12–72 hours for thick preparations to finish. DNase and/or RNase are needed to clean out the remaining cellular components. The downside of this method is that some of the cells could already be destroyed via DNase and/or RNase before even coming into contact with the decellularization agent which will affect the ECM’s integrity [26, 27].
3.6.3. Pressure Gradient
Pressure gradient is commonly used on hollow organs. This mechanism is similar to immersion and agitation. However, it is more optimized by creating a pressure gradient between the extracellular space and the intracellular space, thus optimizing the diffusion process [26, 27].
3.6.4. Supercritical Fluid
This method uses a highly viscous and transportable fluid to kill the cells. This mechanism can also preserve the sample and minimalize the lyophilization process [26, 27].
3.7. Post-Decellularization Evaluation
Post-decellularization evaluation is used to assess the decellularized organs; some of the parameters being measured are the number of DNA strains left, the toxicity, the cellular immunity, and the ECMs [27]. Some of the evaluation methods are discussed as follows.
3.7.1. Histologic and Immunohistochemistry (IHC) Evaluation
Histologic and IHC evaluations are mainly qualitative testing, yet also may serve as a quantitative examination. Qualitative testing is done by comparing the quality of the sample to the original organ, while quantitative testing is done by counting the number of cellular nuclei. First, the organ would need to be fixated in a paraffin block and then cut into smaller pieces. Subsequently, the samples are stained with hematoxylin and eosin for differentiating the ECMs and the nuclei [20, 52]. For a more focused ECM examination, the staining used are toluidine blue and safranin O for GAG assessment, Masson’s trichrome for collagen, and Van Gieson for elastin [20, 53]. IHC examination is used to assess the scaffold’s remaining immunological factors [54, 55]. Other than that, IHC examinations can also evaluate the vascularization potential of the tissue by using anti-CD-31, anti-vWF, and anti-FGF [54]. The downside of this method is that it takes a long time and depends heavily on the examiner [56].
3.7.2. DNA Quantifications
DNA quantification shows the number of DNA in the decellularized graft as a marker for the graft’s immunogenicity ability. The recipient’s immunogenicity tolerance is <50 ng dsDNA/mg of the graft’s dry weight; this number is also the marker of a successful decellularization process. The first step of DNA quantification is to put the sample into an enzymatic solution usually phosphate buffer saline (PBS) for around 24 hours and then the DNA is isolated and examined in a special machine. There are 2 types of DNA quantifier; one of the machine examples is the Quant fluor dsDNA System E2670 Promega that shows the results in DNA/mg scaffold’s dry weight and a nanophotometer that shows the results in nanogram [21].
3.7.3. GAG Quantifications
GAG quantification is used to measure a method’s effectivity in clearing out the cells without destroying the ECMs, thus the need for a control sample for comparison. The GAG quantification is done using a light spectrophotometer [21].
3.7.4. Biomechanics Testing
Biomechanics testing is also quantitative testing that the decellularized trachea is being compared to the control samples. The tracheas will be pulled from 2 sides uniaxially. The data consist of the force given and the increase in length. The proximal, intermediate, and distal parts of the trachea have different biomechanics; thus, testing all 3 of them is recommended [52].
3.7.5. Toxicity Testing
Toxicity testing is done to check on the scaffold’s toxicity level post-decellularization and sterilizing. The purpose is to assess the toxicity level of the tissue induced by the decellularization agents and/or the remaining bacteria on the tissue; thus, toxicity testing is usually done alongside a bacterial load examination to find out if the problem is in the sterilizing process or the decellularization agents [20, 27, 57].
In Table 2, the DNA counts and staining results of various tracheal decellularization methods from 2018 to September 2023 are presented.
4. Conclusions
In conclusion, the selection of a decellularization agent and method should be carefully tailored to the specific tissue or organ under consideration, as each comes with its own set of advantages and disadvantages. According to the findings from the reviewed studies, the optimal scaffold with the minimal DNA content and preserved extracellular matrices (ECMs) is achieved by combining various agents of physical, chemical, and enzymatic nature.
Nonetheless, it is essential to recognize that the quest for the ultimate decellularization method is an ongoing process. Further experiments and research are imperative to explore and refine the selection of agents and methods, aiming for the development of the most effective, safe, and versatile decellularization protocol. The study’s limitations encompass the need for more extensive investigations across various tissue types, focusing on the effects of decellularization on the seeding process and its potential immunogenic effects on the recipient organ. Future research may involve comparative analyses of different decellularization techniques, shedding light on their impacts on tissue biomechanics and immunogenicity, to further advance the field of tissue engineering.
Ethical Approval
Ethical approval is not required in this study as it uses published secondary data sources.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
We would like to express our gratitude to the Department of Thoracic and Cardiovascular Surgery and Tissue Bank of Dr Soetomo General Hospital, Surabaya, Indonesia, for supporting this research.